The present invention relates to a thermostable enzyme which is a DNA polymerase obtainable from Carboxydothermus hydrogenoformans. Furthermore, the present invention relates to the field of molecular biology and provides improved methods for the replication and amplification of deoxyribonucleic (DNA) and ribonucleic acid (RNA) sequences. In a preferred embodiment, the invention provides a method for synthesizing a complementary DNA copy from an RNA template with a thermoreactive DNA polymerase. In another aspect, the invention provides methods for amplifying a DNA segment from an RNA or DNA template using a thermostable DNA polymerase (RT-PCR or PCR).
Heat stable DNA polymerases (EC 2.7.7.7. DNA nucleotidyltransferase. DNA-directed) have been isolated from numerous thermophilic organisms (for example: Kaledin et al. (1980). Biokimiva 45, 644-651; Kaledin et al. (1981) Biokimiva46, 1247-1254; Kaledin et al. (1982) Biokimiya 47, 1515-1521; Ruttimann et al. (1985) Eur. J. Biochem. 149, 41-46; Neuner et al. (1990) Arch. Microbiol. 153, 205-207). For some organisms, the polymerase gene has been cloned and expressed (Lawyer et al. (1989) J. Biol. Chem. 264, 6427-6437; Engelke et al. (1990) Anal. Biochem. 191, 396-400; Lundberg et al. (1991) Gene 108, 1-6; Perler et al. (1992) Proc. Natl. Acad. Sci. USA 89, 5577).
Thermophilic DNA polymerases are increasingly becoming important tools for use in molecular biology and there is growing interest in finding new polymerases which have more suitable properties and activities for use in diagnostic detection of RNA and DNA, gene cloning and DNA sequencing. At present, the thermophilic DNA polymerases mostly used for these purposes are from Thermus species like Taq polymerase from T. aquaticus (Brock et al. (1969) J. Bacteriol. 98. 289-297)
Reverse transcription is commonly performed with viral reverse transcriptases like the enzymes isolated from Avian myeloblastosis virus or Moloney murine leukemia virus, which are active in the presence of Magnesium ions but have the disadvantages to possess RNase H-activity, which destroys the template RNA during the reverse transcription reaction and have a temperature optimum at at 42xc2x0 C. or 37xc2x0 C., respectively.
Alternative methodes are described using the reverse transcriptase activity of DNA polymerases of thermophilic organisms which are active at higher temperatures. Reverse transcription at higher temperatures is of advantage to overcome secondary structures of the RNA template which could result in premature termination of products. Thermostable DNA polymerases with reverse transcriptase activities are commonly isolated from Thermus species. These DNA polymerases however, show reverse transcriptase activity only in the presence of Manganese ions. These reaction conditions are suboptimal, because in the presence of Manganese ions the polymerase copies the template RNA with low fidelity.
Another feature of the commonly used reverse transcriptases is that they do not contain 3xe2x80x2-5xe2x80x2 exonuclease activity. Therefore, misincorporated nucleotides cannot be removed and thus the cDNA copies from the template RNA may contain a significant degree of mutations.
Therefore, it is desirable to develop a reverse transcriptase
which acts at higher temperatures to overcome secondary structures in the template to avoid premature termination of the reaction and to assure the production of cDNA without deletions
which is active in the presence of Magnesium ions in order to prepare cDNA from RNA templates with higher fidelity and
which has 3xe2x80x2-5xe2x80x2-exonuclease in order to remove misincorporated nucleotides before continuation of DNA synthesis and to produce a product with a low mutation frequency.
The present invention adresses these needs and provides a heat stable DNA polymerase active at higher temperatures which has reverse transcriptase activity in the presence of magnesium ions and and which has 3xe2x80x2-5xe2x80x2 exonuclease activity.
It is an object of this invention to provide a polymerase enzyme (EC 2.7.7.7.), characterised in that it has reverse transcriptase activity in the presence of magnesium ions as well as in the presence of manganese ions. In another aspect the invention comprises a DNA polymerase isolated from Carboxydothermus hydrogenoformans (Deutsche Sammiung von Mikroorganismen und Zellkulturen GmbH, Mascheroder Weg 1b, D-38124 Braunschweig, DSM No. 8979). In a further aspect the invention comprises a DNA polymerase having reverse transcriptase activity in the presence of magnesiums ions and in the substantial absence of manganese ions. In a further aspect the invention comprises a DNA polymerase having a molecular mass of about 100 to 105 kDa as determined by in situ PAGE analysis. In a further aspect the invention comprises a reverse transcriptase which is thermostable. In a further aspect the invention comprises a DNA polymerase having 3xe2x80x2-5xe2x80x2-exonuclease activity. In a further aspect the invention comprises a recombinant DNA sequence that encodes DNA polymerase activity of the microorganism Carboxidothermus hydrogenoformans. In a related aspect the DNA sequence is depicted as SEQ ID No. 7. In a second related aspect the invention comprises a recombinant DNA sequence that encodes essentially amino acid residues 1 to 831. In a further aspect the invention comprises a recombinant DNA plasmid that comprises the DNA sequence of the invention inserted into plasmid vectors and which can be used to drive the expression of the thermostable DNA polymerase of Carboxydothermus hydrogenoformans in a host cell transformed with the plasmid. In a further aspect the invention includes a recombinant strain comprising the vector pDS56 carrying the Carboxydothermus hydrogenoformans DNA polymerase gene and designated pAR 4. The E.coli strain (BL21 DE3)pUBS520) carrying the plasmid pAR4 was deposited on the Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH, Mascheroder Weg 1b, D-38124 Braunschweig DSM No. 11179) and is designated AR96.
In referring to a peptide chain as being comprised of a series of amino acids xe2x80x9csubstantially or effectivelyxe2x80x9d in accordance with a list offering no alternatives within itself, we include within that reference any versions of the peptide chain bearing substitutions made to one or more amino acids in such a way that the overall structure and the overall function of the protein composed of that peptide chain is substantially the same asxe2x80x94or undetectably different toxe2x80x94that of the unsubstituted version. For example it is generally possible to exchange alanine and valine without greatly changing the properties of the protein, especially if the changed site or sites are at positions not critical to the morphology of the folded protein.
The DNA polymerase is xe2x80x9cthermostablexe2x80x9d meaning that it is stable to heat and preferentially active at higher temperatures, especially the high temperatures used for denaturation of DNA strands. More particularly, the thermostable DNA polymerases are not substantially inactivated at the high temperatures used in polymerase chain reactions.
The term xe2x80x9creverse transcriptasexe2x80x9d describes a class of polymerases characterized as RNA-dependent DNA polymerases. All known reverse transcriptases require a primer to synthesize a DNA transcript from an RNA template. Historically, reverse transcriptase has been used primarily to transcribe mRNA into cDNA which can then be cloned into a vector for further manipulation.
Other definitions are used in a manner consistent with the art.
Carboxydothermus hydrogenoformans was isolated from a hot spring in Kamchatka by V. Svetlichny. A sample of C. hydrogenoformans was deposited on the Deutsche Sammlung von Mikroorganismen und Zelikulturen GmbH (DSM) under the terms of the BudaPest Treaty and received Accession Number DSM 8979. The thermostable polymerase isolated from Carboxydothermus hydrogenoformans has a molecular weight of 100 to 105 KDa and retains more than 60% of its initial activity after heating to 95xc2x0 C. for 30 minutes. The thermostable enzyme possesses a 5xe2x80x2-3xe2x80x2 polymerase activity, a 3xe2x80x2-5xe2x80x2-exonuclease activity, a 5xe2x80x2-3xe2x80x2-exonuclease activity and a reverse transcriptase-activity which is Mg++-dependent. The polymerase according to the present invention has reverse transcriptase activity in the presence of magnesium ions and in the substantial absence of manganese ions. The thermostable enzyme may be native or recombinant and may be used for first- and second-strand cDNA synthesis, in cDNA cloning, DNA sequencing, DNA labeling and DNA amplification.
For recovering the native protein C.hydrogenoformans may be grown using any suitable technique, such as the technique described by Svetlichny et al. (1991) System. Appl. Microbiol., 14, 205-208. After cell growth one preferred method for isolation and purification of the enzyme is accomplished using the multi-step process as follows:
The cells are thawed, suspended in buffer A (40 mM Tris-HCl, pH 7.5, 0.1 mM EDTA, 7 mM 2-mercaptoethanol. 0.4 M NaCl, 10 mM Pefabloc) and lysed by twofold passage through a Gaulin homogenizer. The raw extract is cleared by centrifugation, the supernatant dialyzed against buffer B (40 mM Tris-HCl, pH 7.5, 0.1 mM EDTA, 7 mM 2-mercaptoethanol, 10% Glycerol) and brought onto a column filled with Heparin-Sepharose (Pharmacia). In each case the columns are equilibrated with the starting solvent and after the application of the sample washed with the threefold of its volume with this solvent. Elution of the first column is performed with a linear gradient of 0 to 0.5 M NaCl in Buffer B. The fractions showing polymerase activity are pooled and ammonium sulfate is added to a final concentration of 20%. This solution is applied to a hydrophobic column containing Butyl-TSK-Toyopearl (TosoHaas). This time the column is eluted with a falling gradient of 20 to 0% ammonium sulfate. The pool containing the activity is dialysed and again transferred to a column, this time with DEAE-Sepharose (Pharmacia), and eluted with a linear gradient of 0-0.5 M NaCl in buffer B. The fourth column contains Tris-Acryl-Blue. (Biosepra) and is eluted as in the preceding case.
Finally the active fractions are dialyzed against buffer C (20 mM Tris-HCl, pH 7.5, 0.1 mM EDTA, 7.0 mM 2-mercaptoethanol. 100 mM NaCl, 50% Glycerol.
Isolation of recombinant DNA polymerase from Carboxydothermus hydrogenoformans may be performed with the same protocol or with other commonly used procedures.
DNA polymerase activity was measured by incorporation of digoxigenin-labeled dUTP into the sythesized DNA and detection and quantification of the incorporated digoxigenin essentially according to the method described in Hxc3x6lke, H.-J.; Sagner, G; Kessler, C. and Schmitz. G. (1992) Biotechniques 12, 104-113.
Determination of reverse transcriptase activity is performed essentially as described for determination of DNA polymerase activity except that the reaction mixture consists of the components as described by example 3. In situ PAGE analysis of polymerase activity and reverse transcriptase activity was performed essentially according to the method described by Spanos A. and Hxc3xcbscher U. ((1983) Methods in Enzymology 91, 263-277). Some minor, but essential modifications to the original method are, that the renaturation of the SDS-denatured polypeptides is performed in the presence of magnesium ions (3 mM) and dATP (0.5-1 xcexcM) to assist refolding.
3xe2x80x2-5xe2x80x2 exonuclease activity is commonly referred as xe2x80x9cproofreadingxe2x80x9d or xe2x80x9ceditingxe2x80x9d activity of DNA polymerases. It is located in the small domain of the large fragment of Type A polymerases. This activity removes mispaired nucleotides from the 3xe2x80x2 end of the primer terminus of DNA in the absence of nucleoside triphosphates (Kornberg A. and Baker T. A.(1992) DNA Replication W. H. Freemann and Company, New York). This nuclease action is suppressed by deoxynucleoside triphosphates if they match to the template and can be incorporated into the polymer.
The 3xe2x80x2- 5xe2x80x2 exonuclease activity of the claimed DNA polymerase can be measured as degradation or shortening of a 5xe2x80x2-digoxygenin-labeled oligonucleotide annealed to template DNA in the absence or presence of deoxyribonucleoside triphosphates or on DNA fragments in the absence or presence of deoxyribonucleoside triphosphates.
Carboxydothermus hydrogenoformans DNA polymerase is the first DNA polymerase isolated from thermophilic eubacteria with a higher activity in the presence of magnesium ions than in the presence of manganese ions as shown in FIG. 1. Compaired to the DNA polymerase activity the reverse transcriptase activity in the presence of magnesium is relatively high. This is shownxe2x80x94in comparison with DNA polymerases from T.filiformis, A.thermophilum and the most commonly used DNA polymerase for reverse transcription T.thermophilus in FIG. 6. The reverse transcriptase activity in dependence of magnesium is of advantage since the DNA polymerases synthesize DNA with higher fidelity in the presence of magnesium than in the presence of manganese (Beckmann R. A. et al. (1985) Biochemistry 24, 5810-5817; Ricchetti M. and Buc H. (1993) EMBO J. 12, 387-396). Low fidelity DNA synthesis is likely to lead to mutated copies of the original template. In addition, Mn2+ ions have been implicated in an increased rate of RNA degradation, particularly at higher temperatures and this can cause the synthesis of shortened products in the reverse transcription reaction.
The DNA sequence (SEQ ID No.: 7) of Carboxydothermus hydrogenoformans polymerase and the derived amino acid sequence of the enzyme are shown in FIG. 5. The molecular weight deduced from the sequence is 94 348 Da, in SDS polyacrylamide gel electrophoresis however the Carboxydothermus hydrogenoformans polymerase has an electrophoretic mobility higher than E.coli pol I (109 kDa) and a lower mobility than Taq polymerase (94 kDa) and Klenow fragment (76 kDa) as shown in FIG. 2. Comparing the migration properties of Taq and E.coli DNA polymerases with those of Carboxydothermus hydrogenoformans polymerase a molecular weight of 100 to 105 kDa can be deduced. Since the Carboxydothermus hydrogenoformans polymerase isolated from the native strain has the same migration properties as the recombinant enzyme the xe2x80x9cslowerxe2x80x9d migration during SDS gel electrophoresis must rather be a property of the enzyme than a cloning artefact. A possible explanation for this phenomenon could be that this enzyme which is derived from a thermophilic organism has a very stable structure which is not completely unfolded under the standard denaturation conditions used.
The production of a recombinant form of Carboxydothermus hydrogenoformans DNA polymerase generally includes the following steps: chromosomal DNA from Carboxydothermus hydrogenoformans is isolated by treating the cells with detergent e.g. SDS and a proteinase e.g. Proteinase K. The solution is extracted with phenol and chloroform and the DNA purified by precipitation with ethanol. The DNA is dissolved in Tris/EDTA buffer and the gene encoding the DNA polymerase is specifically amplified by the PCR technique using two mixed oligonucleotides (primer 1 and 2). These oligo-nucleotides. described by SEQ ID No.: 1 and SEQ ID No.: 2. were designed on the basis of conserved regions of family A DNA polymerases as published by Braithwaite D. K. and Ito J., 1993, Nucl. Acids Res. Vol. 21, p. 787-802. The specifically amplified fragment is ligated into an vector, preferably the pCR(trademark) II vector (Invitrogen) and the sequence is determined by cycle-sequencing. Complete isolation of the coding region and the flanking sequences of the DNA polymerase gene can be performed by restriction fragmentation of the Carboxydothermus hydrogenoformans DNA with another restriction enzyme as in the first round of screening and by inverse PCR (Innis et al., (1990) PCR Protocols; Academic Press, Inc., p. 219-227). This can be accomplished with synthesized oligonucleotide primers binding at the outer DNA sequences of the gene part but in opposite orientation. These oligonucleotides described by SEQ ID Nos. 3 and 4, were designed on the basis of the sequences which were determined by sequencing of the first PCR product described above. As template Carboxydothermus hydrogenoformans DNA is used which is cleaved by restriction digestion and circularized by contacting with T4 DNA ligase. To isolate the coding region of the whole polymerase gene, another PCR is performed using primers as shown in SEQ ID Nos. 5 and 6 to amplify the complete DNA polymerase gene directly from genomic DNA and introducing ends compatible with the linearized expression vector.
SEQ ID No. 1:
Primer 1: 5xe2x80x2-CCN AAY YTN CAR AAY ATH-3xe2x80x2
SEQ ID No. 2:
Primer 2: 5xe2x80x2-YTC RTC RTG NAC YTG-3xe2x80x2
SEQ ID No. 3:
Primer 3: 5xe2x80x2-GGG CGA AGA CGC TAT ATT CCT GAG C-3xe2x80x2
SEQ ID NO. 4:
Primer 4: 5xe2x80x2-GAA GCC TTA ATT CAA TCT GGG AAT AAT C-3xe2x80x2
SEQ ID NO. 5:
Primer 5: 5xe2x80x2-CGA ATT CAA TCC ATG GGA AAA GTA GTC CTG GTG GAT-3xe2x80x2
SEQ ID NO. 6:
Primer 6: 5xe2x80x2-CGA ATT CAA GGA TCC TTA CTT CGC TTC ATA CCA GTT-3xe2x80x2
The gene is operably linked to appropriate control sequences for expression in either prokaryotic or eucaryotic host/vector systems. The vector preferably encodes all functions required for transformation and maintenance in a suitable host, and may encode selectable markers and/or control sequences for polymerase expression. Active recombinant thermostable polymerase can be produced by transformed host cultures either continuously or after, induction of expression. Active thermostable polymerase can be recovered either from host cells or from the culture media if the protein is secreted through the cell membrane.
It is also preferable that Carboxydothermus hydrogenoformans thermostable polymerase expression is tightly controlled in E.coli during cloning and expression. Vectors useful in practicing the present invention should provide varying degrees of controlled expression of Carboxydothermus hydrogenoformans polymerase by providing some or all of the following control features: (1) promoters or sites of initiation of transcription, either directly adjacent to the start of the polymerase gene or as fusion proteins, (2) operators which could be used to turn gene expression on or off, (3) ribosome binding sites for improved translation, and (4) transcription or translation termination sites for improved stability. Appropriate vectors used in cloning and expression of Carboxydothermus hydrogenoformans polymerase include, for example, phage and plasmids. Example of phage include lambda gtII (Promega), lambda Dash (Stratagene) lambda ZapII (Strata-gene). Examples of plasmids include pBR322, pBTac2 (Boehringer Mannheim), pBluescript (Stratagene), pSP73 (Promega), pET3A (Rosenberg, supra, (1987) Gene 56:125-135), pASK75 (Biometra), pDS56 (Stxc3xcber, D., Matile, H. and Garotta G. (1990) Immunological Methods, Letkovcs, I. and Pernis, B., eds.) and pET11C (Studier, F. W. (1990) Methods in Enzymology, 185:60-89). According to the present invention the use of a plasmid has shown to be advantageously, particularly pDS56. The Plasmid pDS56 carrying the Carboxydothermus hydrogenoformans DNA polymerase gene is then designated pAR4.
Standard protocols exist for transformation, phage infection and cell culture (Maniatis, et al. (1982) Molecular Cloning: A Laboratory Manual, Cold Spring Harbour Laboratory Press). Of the numerous E.coli strains which can be used for plasmid transformation, the preferred strains include JM110 (ATCC 47013), LE392 pUBS 520 (Maniatis et al. supra; Brinkmann et al., (1989) Gene 85:109-114;), JM101 (ATCC No. 33876), XL1 (Stratagene), and RR1 (ATCC no. 31343), BL21 (DE3) pUBS520 (Brinkmann, U. et al. (1989) Gene 85, 109-114) and BL21 (DE3) plysS (Studier, F. W. et al., (1990) Methods in Enzymology, supra). According to the present invention the use of the E. coli strain BL21 (DE3) pUBS520 has shown to be advantageously. The E. coli strain BL21 (DE3) pUBS520 transformed with the plasmid pAR4 is then designated AR96(DSM No. 11179). E.coli strains XL1-Blue (Stratagene), and ER1458 (Raleigh, E. A. et al., (1988) Nucleic Acids Research 16:1563-1575) are among the strains that can be used for lambda phage, and Y1089 can be used for lambda gt11 lysogeny. The transformed cells are preferably grown at 37xc2x0 C. and expression of the cloned gene is induced with IPTG.
Isolation of the recombinant DNA polymerase can be performed by standard techniques. Separation and purification of the DNA polymerase from the E.coli extract can be performed by standard methods. These methods include, for example, methods utilizing solubility such as salt precipitation and solvent precipitation, methods utilizing the difference in molecular weight such as dialysis, ultra-filtration, gel-filtration, and SDS-polyacrylamide gel electrophoresis, methods utilizing a difference in electric charge such as ion-exchange column chromatography, methods utilizing specific interaction such as affinity chromatography, methods utilizing a difference in hydrophobicity such as reversed-phase high performance liquid chromatography and methods utilizing a difference in isoelectric point such as isoelectric focussing electrophoresis.
The present invention provides improved methods for efficiently transcribing RNA and amplifying RNA or DNA. These improvements are achieved by the discovery and application of previously unknown properties of thermoactive DNA polymerases.
The thermostable enzyme of this invention may be used for any purpose in which such enzyme activity is necessary or desired. In a particularly preferred embodiment, the enzyme catalyzes the nucleic acid amplification reaction known as PCR. This process for amplifying nucleic acid sequences is disclosed and claimed in U.S. Pat. No. 4,683,202. The PCR nucleic acid amplification method involves amplifying at least one specific nucleic acid sequence contained in a nucleic acid or a mixture of nucleic acids and produces double-stranded DNA. Any nucleic acid sequence, in purified or nonpurifled form, can be utilized as the starting nucleic acid(s), provided it contains or is suspected to contain the specific nucleic acid sequence desired. The nucleic acid to be amplified can be obtained from any source, for example, from plasmids such as pBR322, from cloned DNA or RNA, from natural DNA or RNA from any source, including bacteria, yeast, viruses, organelles, and higher organisms such as plants and animals, or from preparations of nucleic acids made in vitro.
DNA or RNA may be extracted from blood, tissue material such as chorionic villi, or amniotic cells by a variety of techniques. See, e.g., Maniatis et al., 1982, Molecular Cloning: A Laboratory Manual (Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y.) pp. 280-281. Thus the process may employ, for example, DNA or RNA, including messenger RNA, which DNA or RNA may be single-stranded or double-stranded. In addition, a DNA-RNA hybrid which contains one strand of each may be utilized.
The amplification of target sequences in DNA or from RNA may be performed to proof the presence of a particular sequence in the sample of nucleic acid to be analyzed or to clone a specific gene. DNA polymerase from Carboxydothermus hydrogenoformans is very useful for these processes. Due to its 3xe2x80x2-5 xe2x80x2 exonuclease activity it is able to synthesize products with higher accuracy as the reverse transcriptases of the state of the art.
DNA polymerase from Carboxydothermus hydrogenoformans may also be used to simplify and improve methods for detection of RNA target molecules in a sample. In these methods DNA polymerase from Carboxydothermus hydrogenoformans may catalyze: (a) reverse transcription, (b) second strand cDNA synthesis, and. if desired, (c) amplification by PCR The use of DNA polymerase from Carboxydothermus hydrogenoformans in the described methods would eliminate the previous requirement of two sets of incubation conditions which are necessary due to the use of different enzymes for each step. The use of DNA polymerase from Carboxydothermus hydrogenoformans may be used to perform RNA reverse transcription and amplification of the resulting complementary DNA with enhanced specificity and with fewer steps than previous RNA cloning and diagnostic methods.